The type II collagen fragments Helix-II and CTX-II reveal different enzymatic pathways of human cartilage collagen degradation  N. Charni-Ben Tabassi, Ph.D., S. Desmarais, Ph.D., A.-C. Bay-Jensen, J.M. Delaissé, Ph.D., M.D. Percival, Ph.D., P. Garnero, Ph.D., D.Sc.  Osteoarthritis and Cartilage  Volume 16, Issue 10, Pages 1183-1191 (October 2008) DOI: 10.1016/j.joca.2008.02.008 Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 Efficiency of Cat (A) and MMPs (B) to solubilize human articular cartilage collagen in vitro human cartilage powder was incubated for 24h at 37°C with 1μM of activated recombinant human enzymes (see Materials and methods for details). The level of collagen solubilization was evaluated by measuring hydroxyproline in the supernatant of each incubation mixture and was expressed as a percentage of the amount of collagen present initially in the tube. The bars are the mean+SD of two different incubation experiments performed in duplicate. Osteoarthritis and Cartilage 2008 16, 1183-1191DOI: (10.1016/j.joca.2008.02.008) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 Release of the type II collagen fragments Helix-II (A) and CTX-II (B) by Cats and MMPs from human cartilage collagen. Cartilage powder was incubated as described in Fig. 1 and Helix-II and CTX-II were measured in the supernatant by ELISA. Helix-II and CTX-II values were expressed in nmol/μg of hydroxyproline released in the supernatant. The bars are the mean+SD of two different incubation experiments performed in duplicate. Osteoarthritis and Cartilage 2008 16, 1183-1191DOI: (10.1016/j.joca.2008.02.008) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 Effects of MMPs in releasing Helix-II (A) and CTX-II (B) from soluble Cat K generated fragments. Fragments generated by the action of Cat K on cartilage powder as described in Fig. 2 were submitted to a second digestion with MMPs for 24h at 37°C (see Materials and methods for details). The bars are the mean+SD of different two incubation experiments performed in duplicate. The horizontal dotted line in panel A indicates the basal level of Helix-II resulting from the initial Cat K digestion and no subsequent digestion with MMP (control no MMP). Basal CTX-II value is equal to 0 in panel B, as Cat K cannot generate CTX-II immunoreactivity from insoluble cartilage matrix (see Fig. 2). Osteoarthritis and Cartilage 2008 16, 1183-1191DOI: (10.1016/j.joca.2008.02.008) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 Effects of Cats in releasing Helix-II (A and B) and CTX-II (C and D) from soluble MMP-7 (A and C) or MMP-13 (B and D) generated fragments. Fragments generated by the action of MMP-7 or MMP-13 on cartilage powder as described in Fig. 2 were submitted to a second digestion with Cats for 24h at 37°C. The bars are the mean+SD of two incubation experiments performed in duplicate. In each panel, the horizontal dotted line indicates the basal level of Helix-II or CTX-II observed during the initial degradation with MMP and no subsequent Cat digestion (Control no Cat). Osteoarthritis and Cartilage 2008 16, 1183-1191DOI: (10.1016/j.joca.2008.02.008) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 Comparative effect of a cysteine proteinase inhibitor (E-64) (A, C) and an MMP inhibitor (GM6001) (B, D) on the release of Helix-II (A, B) and CTX-II (C, D) in culture of human OA cartilage explants. Human cartilage sections were incubated in a buffer favorable to either cysteine proteases or MMPs activity and in the presence (filled circle) or absence (open circle) of 50-μM E-64 or 10-μM GM6001 for 12, 36, 60 and 84h. All cartilage explants were initially pre-incubated for 12h in absence of inhibitors to activate endogenous enzymes and then further incubated for up to 72h (total incubation time, 84h) with or without protease inhibitors. At each time point, collagen content within the cartilage explant was estimated by hydroxyproline determination. Supernatant was also analyzed at each time point and conditions for release of hydroxyproline, Helix-II and CTX-II (see Materials and methods for details). Levels of Helix-II and CTX-II were corrected for total collagen content estimated from the sum of hydroxyproline within the cartilage explants and the supernatant. Helix-II and CTX-II values after 36, 60 and 84h of incubation were then expressed in percent of the values of the 12h activation period. Paired cartilage explants from the same patient were used in each incubation condition (for example, MMP buffer with or without inhibitor and/or APMA). Each bar represents the mean value+SD of duplicate incubations from three consecutive cartilage sections taken vertically from the same cartilage explant. The P values analysis of variance (ANOVA) refer to the difference in the time dependent changes of Helix-II and CTX-II release between experiments performed with and without enzyme inhibitors. Osteoarthritis and Cartilage 2008 16, 1183-1191DOI: (10.1016/j.joca.2008.02.008) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 6 Schematic representation of a type II collagen molecule and localization of the sequence of the biochemical markers Helix-II and CTX-II and of the enzymatic cleavage sites involved in their generation. In bold italic font are the sites determined in the present study. For Cat B in the Helix-II sequence, the exact site was not determined. 1Site reported by Billinghurst et al.36; 2sites reported by Lohmander et al.37 Osteoarthritis and Cartilage 2008 16, 1183-1191DOI: (10.1016/j.joca.2008.02.008) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions